Enzymatic oxidation of hydrocarbons



United States Patent 3,326,771 ENZYMATIC OXIDATION OF HYDROCARBONS Richard I. Leavitt, Pennington, N.J., assignor to Mobil Oil Corporation, a corporation of New York No Drawing. Filed Dec. 12, 1963, Ser. No. 329,970 15 Claims. (Cl. 195-3) This invention relates to the enzymatic oxidation of hydrocarbons, and involves both the preparation of enzymes from microorganisms and, in particular, the use of the same to oxidize hydrocarbons. It further relates to a method of examining and evaluating the activity of the enzymes, whether contained in the cells of the microorganisms or existing in the cell-free state, in a hydrocarbon oxidation system.

An advantage of the invention resides in providing a convenient and simplified way of preparing useful oxygenated hydrocarbons, particularly oxygenated aromatics, in good yield. In respect of the determination of enzymic activity, the invention eliminates the need of making tedious gas measurements and gas analyses, as heretofore practiced. Other advantages will be apparent from the ensuing description.

In brief, the invention comprises mixing an enzyme of a microorganism that is a hydrocarbon oxidizer, as described hereinafter, with the hydrocarbon to be oxidized, adding to the mixture a tetrazolium compound as an electron acceptor, a sulfhydryl group-containing compound to enhance the activity of the enzyme, and an at least partially water-miscible alkanol to improve the compatibility of the foregoing components, and then incubating the mixture to oxidize the hydrocarbons. At this point the oxygenated hydrocarbon products may be recovered from the mixture, or, if the enzyme activity is under study, an examination of the mixture may be made, as described herein, to evaluate such activity. Even though enzyme activity is being studied, oxygenated hydrocarbon products may be recovered as the amount of the mixture required for the study is negligible.

The invention further comprises the microbiological oxidation of a hydrocarbon starting with the bacterial cells; and, whether whole cells or cell-free enzymes are used, it comprises a procedure for evaluating enzymic activity.

Considering the invention in detail, and in a more or less orderly way in respect of each component feature thereof, the microorganisms with which it is useful are preferably those from the genera consisting of Pseudomonas and Nocardia, including such species as Ps. aeruginosa, Ps. oleovorans, Ps. putida, Ps. fluorescens, Ps. boreopolis, Ps. methan 'ca, N. corallinus, N. opacus, N. paralfinae, N. salmonicolor. Most of the Pseudomonas are pigment producers and these comprise a preferred class. Another suitable genus of hydrocarbon-oxidizing microbes with which the invention may be practiced is Bacillus, which includes such species as B. hexacarbovorum, B. mesentericus, B. toluolicum, etc. Hydrocarbon oxidizers from the genus Mycobacterium are useful, including M. phlei, M. rubrum, M. luteum, M. lactz'cola, M. album, M. byalinicum, and M. leprae.

A wide variety of hydrocarbons may be oxidized according to the invention. These comprise both cyclic and alicyclic hydrocarbons, the former including benzene,

naphthalene, anthracene, phenanthrene, other polynuclear aromatics, and particularly alkyl-substituted aromatics, of

which representative examples include compounds like toluene, xylene, ethylbenzene, n-propylbenzene, cumene, pseudocumene, cymene, and the like; and the alicyclic hydrocarbons comprise saturated and unsaturated 5- or 6-membered cycloparatiins having 1, 2, or more rings,

these also being preferably substituted by alkyl groups.

compound which is soluble in the mixture,

Also included are aliphatic hydrocarbons comprising saturated and unsaturated compounds including straight and branched chain alkenes having up to 30 or more carbon atoms and straight and branched chain alkenes having 2 to 16 or more carbon atoms. Alkyl-substituted aromatics are of particular interest because they are easier to work with in an enzyme system.

Cells of the microorganism may be grown in an agitated system using as the sole source of carbon the hydrocarbon which is to be oxidized. In addition to the hydrocarbon, the system or culture also contains a medium comprising conventional mineral salts, including a source of nitrogen such as sodium nitrate or ammonium sulfate. After a suitable period of growth, the cells "are harvested, washed, and then suspended in an aqueous medium and the cells ruptured to free or make available intracellular enzyme components thereof. Cell rupture may be done by exposing the cells to high frequency oscillations or by other conventional methods including grinding in the presence of abrasives, shaking with "abrasives, exposure to lysozyme, compressing and release of pressure as in a French pressure cell, or other physical-chemical treatments designed 2o rupture the cell and release soluble components there- The tetrazolium salt is a stable, water-soluble, organic salt, functioning as a cationic indicator which undergoes reduction as the hydrocarbon is oxidized and which does not readily reoxidize in the presence of oxygen. In its oxidized state the salt is pale or colorless, but it becomes colored and water insoluble as it is reduced. Suitable tetrazolium salts include 2,3,S-triphenyltetrazolium chloride, 2,3-diphenyl-5-methyl tetrazolium chloride, 2,5-diphenyl-3-(4-styrylphenyl) tetrazolium chloride, thiazoyl blue, tetrazolium violet, iodonitrotetrazolium chloride, etc. Ditetrazolium salts are suitable, such as neotetrazolium, n-nitroblue tetrazolium, p-nitroblue tetrazolium, m-nitroviolet tetrazolium, violet tetrazolium (toluidine), blue tetrazolium, blue tetrazolium (p-anisyl), blue tetrazolium (piperonyl), blue tetrazolium (veratryl), 3,3'-dianisole bis-4,4'-(3,5-diphenyl) diformazan, etc. As indicated, the tetrazolium salts function as electron acceptors in the oxidation of the hydrocarbon. During such oxidation, electrons are released, and the electron acceptor, as the name implies, takes the released electrons.

The alkanol helps to bring into intimate contact the various components involved, that is, the hydrocarbon to be oxidized, the cell extract comprising the enzyme material, the tetrazolium salt, and the sulfhydryl-containing compound, and for this reason it is regarded as a contact-improving or contact-enhancing agent. In other words, it increases the compatibility or miscibility of the components. As water is present, the alkanol is water miscible at least to some extent. Preferably the alkanol is a low molecular weight alcohol, particularly one having 2 to 4 carbon atoms, and including ethanol, propanol, isopropanol, l-butanol, 2-butanol, isobutanol, and tertbutanol. Also useful are pentanols like 2-pentanol, and hexanols like 2,3-dimethyl-2-butanol. As illustrated in Example 3, enzyme activity for oxidizing hydrocarbons increases as the number of carbon atoms in the alcohol increases within the range of 2 to 4 carbon atoms.

The sulhydryl-containing compound enhances the enzyme activity. It may be added per se to the mixture, in which case it may be any sulfhydryl-containing organic although preferably it is a low molecular weight aliphatic compound.

Preferably, too, it contains a carboxyl group in addition acids, Z-arninobenzenethiol, and the like. Another preferred class of sulfhydryl-containing compounds are those having a hydroxy group in addition to the sulfhydryl group, it having been found that in this case the compound may function as both the contact-enhancing and the enzyme activity-enhancing agent. Illustrative of this last-mentioned class are the mercaptoalkanols such as mercaptoethanol, 2,3 dimercaptopropanol, 3-mercapto- 1,2-propanediol, etc.

The procedure for microbially oxidizing a hydrocarbon is illustrated in the examples below but may be described briefly as follows. The selected microorganism is grown in a culture medium comprising the mineral salts, nitrogen source, and, as the sole source of carbon, the desired hydrocarbon. After the cells are grown, the soluble enzyme extract is removed from them as described. The bacterial extract comprising the enzyme or enzymes is then mixed with the hydrocarbon to be oxidized, tetrazolium salt, sulfhydryl compound, and alkanol, and the mixture is incubated. Incubation may be carried out at about 20 to about 55 C., preferably around 30 C., and for times generally extending up to about an hour. During incubation, the mixture is preferably stirred and may be kept under a nitrogen atmosphere At the conclusion of the oxidation, a small amount of strong mineral acid may be added to stop the reaction. The oxygenated hydrocarbon products are recovered, suitably by extraction with a solvent such as ether. After evaporation of the ether from the extract, the oxygenated products are separable from each other by conventional procedures such as distillation, fractional crystallization, and the like.

It may be noted that the hydrocarbon upon which the cells are initially grown is preferably the same as that which is incubated with the bacterial extract, although it may be a different hydrocarbon. For example, if p-cymene is used for cell growth, it is preferably used in the step of incubating the bacterial extract mixture, although in this step a different hydrocarbon such as ethylbenzene, propylbenzene, and the like may also be employed. Desirably, such different hydrocarbon is a member of the same homologous series as, or is chemically similar to, the first used hydrocarbon.

Oxygenated hydrocarbon products include alcohols, aldehydes, ketones, and acids. A hydrocarbon like p-cymene may yield products such as p-isopropyl benzaldehyde and p-isopropyl benzoic acid, the former being of value in perfumery. Other products are hydroperoxides, which are of particular interest as intermediates. Most of the other oxygenated products also have utility as intermediates.

To study the activity of an enzyme extracted from the microorganisms, essentially the foregoing procedure is employed, that is, the selected cells are grown, then ruptured to free the soluble enzyme material, the latter is then mixed with the hydrocarbon to be studied and also with the tetrazolium salt, alkanol, and sulfhydryl compound, and the whole is incubated. However, instead of, or in addition to, recovering the hydrocarbon oxidation products, a small amount of the mixture is subjected to a suitable test to quickly determine the extent of oxidation and thus to provide information on the enzyme activity. Suitably, the sample of mixture may be subjected to a spectrophotometric test such as one involving the selective adsorption of infrared rays, or one involving the absorption of visible light rays of a suitable wave length. As indicated above, the tetrazolium salt is reduced as the hydrocarbon is oxidized, and in addition acquires a distinctive color, which in the foregoing tests will adsorb radiation, the amount of which is measured. By comparing the adsorbancy of the mixture with that of a control, the extent of reduction of the tetrazolium salt may be obtained, which in turn corresponds to the extent of oxidation of the hydrocarbon and in turn to the enzyme activity.

It will be understood that during microbial oxidation, the mixture is accessible to atmospheric oxygen.

The amounts of the various components may be given on the basis of 1 ml. of the mixture which is incubated. Thus, per ml. of such mixture, there may be present the following: (1) bacterial extract, 0.1 to 60, preferably 3 to 10 mg. of protein; (2) hydrocarbon, 0.1 to 30, preferably 10 to 30 micromoles; (3) tetrazolium salt, 0.1 to 10, preferably 1 to 5 micromoles; (4) alkanol, 1 to 100, preferably 10 to 50 micromoles; and (5) sulfhydryl compound, 0.3 to 30, preferably 10 to 30 micromoles. As is apparent the amount of the bacterial extract is given in terms of its protein content. It will be understood that in each ml. of the foregoing mixture, water is also present. A suitable method of bringing together all components is to first dissolve or disperse each in water and then to combine these aqueous portions. Thus, the bacterial extract, tetrazolium salt, alkanol, and sulfhydryl compound are each dissolved in water, the hydrocarbon is suitably dispersed or emulsified in water (although it can also be added directly), and the resulting aqueous portions are combined. In practice, large amounts of each aqueous portion may be made up, and aliquot portions then taken and mixed.

The invention may be illustrated by the following examples:

Example 1 An organism identified as Pseudomonas aeruginosa was grown in an agitated system on a mineral salts media using ammonium sulfate as the sole source of nitrogen and p-cymene as the sole source of carbon. After 72 hours, the cells were harvested by centrifugation, yield about 12 g., washed with a 0.02 molar aqueous solution of phosphate buffer of pH 7.0 and then suspended in a 0.05 molar aqueous solution of phosphate of pH 7.0 to provide a concentration of 1 g. of cells per 4 ml. of suspension. This suspension was then exposed to the high frequency oscillations of an ultrasonic device identified as a Branson sonofier to rupture the cells and to free soluble cell components, including enzymes, which then dissolved in the phosphate solution. Cell debris was removed from the solution by centrifugation at 37,000 times g.

The clear solution was then tested for its ability to oxidize p-cymene. For this purpose 5 mixtures were prepared as follows:

No. 1: In each ml., this solution contained- Bacterial extract ml 0.2 Tris buffer, pH 8.4 micromoles p-Cymene do 10 2,3,S-triphenyltetrazolium chloride d0 2 No. 2: Same as No. 1 plus 30 micromoles cysteine.

No. 3: Same as No. 1 plus 30 micromoles ethanol.

No. 4: Same as No. 1 plus 30 micromoles each of cysteine and ethanol.

No. 5: Same as No. 1 plus 30 micromoles mercaptoetha- Tris is tris-(hydroxymethyl-aminomethane). Each mixture was incubated for 15 minutes under nitrogen at 25 C., with shaking. In each case the oxidation of the hydrocarbon was stopped by the addition of 0.1 ml. of 3 N sulfuric acid. Each mixture was then tested for adsorption of visible light rays of a wave length of 500 millimicrons (0.5 mi- All values were corrected for endogenous reduction of the tetrazolium compound. The value of 0.30 for No. 5 represents a 30% reduction in the transmission of light owing to the presence of the colored form of the tetrazolium compound; and similarly, the value of 0.00 for No. 1 represents no reduction in light transmission, or complete transmission owing to the absence of the colored compound. It will be seen that the presence of both sulfhydryl and alcoholic hydroxyl groups, as in Nos. 4 and '5, results in the formation of appreciable amounts of the reduced colored compound, as indicated by the considerable adsorption of light, which in turn denotes thepresence of considerable amounts of oxidized hydrocarbons. Mixtures Nos. 4 and 5 also represent a good level of enzyme activity. In the presence of cysteine alone, or ethanol alone, as in Nos. 2 and 3, a low level of enzyme activity was observed. As is apparent, No. 4 shows almost a 9-fold greater activity than No. 3.

Example 2 The work of Example 1 was repeated, except that the organism was Nocardia salmom'color, strain 107-332, and n-tetradecane was used as the sole source of carbon instead of p-cymene. Two mixtures, identified as Nos. 6 and 7, were prepared as follows:

Inother words, No. 6 was the control; while No. 7 contained cysteine and ethanol. Light adsorption tests as described in Example 1 showed the following results:

Mixture No.: Adsorbency 6 0.00 7 .50

It is apparent that No. 7 indicates the presence of a con sidera-ble amount of oxidized hydrocarbons as well as a high level of enzyme activity.

Example 3 The work of Example 1 was repeated, except that in preparing the mixtures, identified here as Nos. 8, 9, 10, and 11, certain changes were made as follows:

No. 8: Same as No. 1 plus micromoles cysteine.

No. 9: Same as No. 8 plus 10 micromoles ethanol. No. 10: Same as No. 8 plus 10 micromoles propanol. No. 11: Same as No. 8 plus 10 micromoles n-butanol.

Thus, No. 8, which contained cysteine but no alkanol, served as the control. Light adsorption tests showed the following:

Mixture No.: Adsorbency 8 0.03

As is apparent, the use of propanol and n-butanol results in considerably increased amounts of oxidized hydrocarbons and high levels of enzyme activity.

The tetrazolium salt is of particular value in studying enzyme activity as it provides a measure of convenience in the assay. However, when the effort is to produce oxygenated hydrocarbon products, the salt may be omitted from the mixture to be incubated.

In some cases, whether enzyme activity is under study or whether oxygenated products are produced, it is possible to omit the alkanol or contact-improving agent. Although such omission tends to reduce the level of enzyme activity, a workable level of activity is still obtainable. Preferably, however, the alkanol is present.

It will be understood that the invention is capable of obvious variations without departing from its scope.

In the light of the foregoing description, the following is claimed.

1. Method for microbiologically oxidizing a hydrocarbon which comprises growing in a fermentation mixture in which a hydrocarbon is present as the sole source of carbon a microorganism that is a hydrocarbon oxidizer and is selected from the class consisting of hydrocarbonoxidizing species of Pseudomonas, Nocardia, Bacillus, and Mycobacterium, said mixture containing in addition to said hydrocarbon a medium capable of supporting growth of said microorganism, harvesting and washing the cells of the microorganism, suspending the cells in an aqueous medium and rupturing the same, thereby dissolving soluble enzymes thereof in said aqueous medium, then adding a hydrocarbon to be oxidized to the aqueous medium together with a tetrazolium salt as an electron acceptor and an organic agent having an enzyme activityenhancing effect and a compatibility-improving action on the other components, said agent being at least partially miscible with water and being characterized by the presence of sulfhydryl and alcoholic OH groups, incubating said aqueous medium, and forming oxygenated hydrocarbons therein.

2. Method of claim 1 wherein said agent comprises a mixture of a low molecular weight alkanol and a sulfhydryl-containing compound.

3. Method of claim 1 wherein said agent comprises an organic compound containing both sulfhydryl and hydroxy groups.

4. Method for microbiol-ogically oxidizing a hydrocarbon which comprises growing in a fermentation mixture in which said hydrocarbon is present as thev sole source of carbon a microorganism that is a hydrocarbon oxidizer and is selected from the class consisting of hydrocarbon-oxidizing species of Pseudomonas, Nocardia, Bacillus, and Mycobacterium, said mixture containing in addition to said hydrocarbon a nutrient medium capable of supporting growth of said microorganism, harvesting and washing the cells of the microorganism, suspending the cells in an aqueous medium and rupturing the same, thereby dissolving at least one soluble enzyme thereof in said aqueous medium, then adding a hydrocarbon to be oxidized to the aqueous medium together with a tetrazolium salt as an electron acceptor, an organic sulfhydrylcontaining compound, which is at least partially miscible with water, to enhance the activity of said enzyme, and a low molecular weight alkanol, said salt being reduced as said hydrocarbon is oxidized, said alkanol being at least partially miscible with water, and forming oxygenated products of said hydrocarbon.

5. Method of claim 4 in which said firstand secondmentioned hydrocarbons are the same.

-6. Method of claim 4 in which said firstand secondmentioned hydrocarbons are diiferent.

7. Method of claim 4 in which said firstand secondmentioned hydrocarbons are alkyl-substituted aromatics.

8. Method for determining the activity of an enzyme of a microorganism in the enzymic oxidation of a hydrocarbon, said microorganism being a hydrocarbon oxidizer selected from the class consisting of hydrocarbon-oxidizing species of Pseudomonas, Nocardia, Bacillus, and Mycobacterium, which comprises oxidizing the hydrocarbon by incubating the same with said enzyme in the presence of a tetrazolium salt as an electron acceptor, a low molec ular weight alkanol, and an organic sulfhydryl-containing compound which is at least partially miscible with water, said salt being reduced as said hydrocarbon is oxidized, and measuring the amount of tetrazolium salt reduced, such amount corresponding to the amount of hydrocarbon that is oxidized and being a measure of the activity of said enzyme.

9. Method for enzymically oxidizing a hydrocarbon with an enzyme of a microorganism that is a hydrocarbon oxidizer and is selected from the class consisting of hydrocarbon-oxidizing species of Pseudomonas, Nocardia, Bacillus, and Mycobacterium, which comprises oxidizing the hydrocarbon by incubating the same with said enzyme in the presence of a tetrazolium salt as an electron acceptor and in the presence of a low molecular weight alkanol and an organic sulfhydryl-containing compound, said salt being reduced as said hydrocarbon is oxidized, said alkanol being at least partially miscible with water and having a compatibility-enhancing effect on the other components, said sulfhydryl compound being at least partially miscible with Water and serving to enhance the activity of said enzyme, and obtaining oxygenated hydrocarbons in the incubated mixture.

10. Method of claim 9 in Which said microorganism is selected from hydrocarbon-oxidizing species of Pseudomonas.

11. Method of claim 9 in Which said microorganism is selected from hydrocarbon-oxidizing species of Nocardia.

12. Method of claim 9 in which said hydrocarbon is an alkyl-substituted aromatic.

13. Method for enzymically oxidizing a hydrocarbon with an enzyme of a microorganism that is a hydrocarbon oxidizer and is selected from the class consisting of hydrocarbon-oxidizing species of Pseudomonas, Nocardia, Bacillus, and Mycobacterium, which comprises oxidizing the hydrocarbon by incubating the same with said enzyme in the presence of a tetrazoliurn salt as an electron acceptor and in the presence of an organic agent having an enzyme activity-enhancing efiect and a compatibility-improving action on the other components, said agent being at least partially miscible with water and being characterized by the presence of sulfhydryl and alcoholic --OH groups, and obtaining oxygenated hydrocarbons in the incubated mixture.

14. Method for enzymically oxidizing a hydrocarbon with an enzyme of a microorganism that is a hydrocarbon oxidizer and is selected from the class consisting of hydrocarbon-oxidizing species of Pseudomonas, Nooardia, Bacillus, and Mycobacterium, which comprises oxidizing the hydrocarbon by incubating same with said enzyme in the presence of a low molecular Weight alkanol and an organic sulfhydryl-containing compound, said alkanol being at least partially miscible with water and having a compatibility enhancing efiect on the other components, said sulfhydryl compound being at least'partially miscible With water and serving to enhance the activity of said enzyme, and obtaining oxygenated hydrocarbons in the incubated mixture.

15. Method for enzymically oxidizing a hydrocarbon with an enzyme of a microorganism that is a hydrocarbon oxidizer and is selected from the class consisting of hydrocarbon-oxidizing species of Pseudomonas, Nocardia, Bacillus, and Mycobacterium, which comprises oxidizing the hydrocarbon by incubating the same with said enzyme in the presence of an organic sulfhydryl-containing compound which is at least partially miscible with water as an enxyme activity-enhancing agent, and obtaining oxygenated hydrocarbons in the incubated mixture.

References Cited UNITED STATES PATENTS 2,697,062 12/1954 Cramer 1953 3,057,784 10/1962 Davis et al. 19528 3,212,993 10/1965 Hitzman l28 A. LOUIS MONACELL, Primary Examiner.

D. M. STEPHENS, A. E. TANENHOLTZ,

Assistant Examiners. 

15. METHOD FOR ENZYMICALLY OXIDIZING A HYDROCARBON WITH AN ENZYME OF A MICROOGANISM THAT IS A HYDROCARBON OXIDIZER AND IS SELECTED FROM THE CLASS CONSISTING OF HYDROCARBON-OXIDIZING SPECIES OF PSEUDOMONAS, NOCARDIA, BACILLUS, AND MYCOBACTERIUM, WHICH COMPRISES OXIDIZING THE HYDROCARBON BY INCUBATING THE SAME WITH SAID ENZYME IN THE PRESENCE OF AN ORGANIC SULFHYDRYL-CONTAINING COMPOUND WHICH IS AT LEAST PARTIALLY MISCIBLE WITH WATER AS AN ENXYME ACTIVITY-ENHANCING AGENT, AND OBTAINING OXYGENATED HYDROCARBONS IN THE INCUBATED MIXTURE. 